Rapid-cycle PCR method to detect Bordetella pertussis that fulfills all consensus recommendations for use of PCR in diagnosis of pertussis

No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of Bordetella pertussis infections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene (Bordetella spp.). In specimens positive for Bordetella spp., B. pertussis was differentiated from Bordetella parapertussis and Bordetella bronchiseptica by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse beta-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS481 and IS1001 PCR ( approximately 1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS481 and IS1001. Fifty-one specimens were positive for B. pertussis by POR and IS481 PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS481 PCR. A total of 652 specimens were negative by both methods. B. parapertussis was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of B. pertussis.     

Evaluation of real-time PCR for detection of and discrimination between Bordetella pertussis,Bordetella parapertussis,and Bordetella holmesii for clinical diagnosis

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.     

Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples.

Several genes and sequences in Bordetella pertussis have been used as targets in diagnostic PCR assays. A previously developed single-step PCR assay for the detection of B. pertussis was based on an insertion sequence, IS480, that is present in about 70 to 80 copies in each genome. The diagnostic sensitivity, specificity, and reliability of this assay with aspirated and heat-treated samples from the nasopharynx of patients and their contacts was improved by the use of a nested PCR configuration. The nested PCR assay produced a 205-bp fragment with all of the 115 B. pertussis strains tested and was negative with all strains belonging to other Bordetella species (n = 44) as well as other bacteria commonly found in the upper respiratory tract (n = 115). The diagnostic value of the assay was verified by giving positive results for B. pertussis in all the 51 nasopharyngeal aspirates from culture-positive patients. The assay also detected 18 positive aspirates from a total of 196 culture-negative patients. A confirmatory cleavage of the 205-bp nested PCR product by MvaI gave in all cases two bands of 88 and 117 bp. In conclusion, this newly developed nested PCR assay was shown to be reasonably fast and uncomplicated, with an optimal sensitivity and a high degree of specificity for the diagnosis of B. pertussis in aspirated nasopharyngeal samples processed simply by heat treatment. The detection level in the nested PCR was about 10 bacteria per ml, or seven to eight insertion sequence copies per 10 microliters of boiled sample.     

Insertion sequences shared by Bordetella species and implications for the biological diagnosis of pertussis syndrome

The molecular diagnosis of pertussis and parapertussis syndromes is based on the detection of insertion sequences (IS) 481 and 1001, respectively. However, these IS are also detected in the genomes of various Bordetella species, such that they are not specific for either B. pertussis or B. parapertussis. Therefore, we screened the genome of recently circulating isolates of Bordetella species to compare the prevalence of IS481, IS1001 and, also IS1002 with previously published data and to sequence all IS detected. We also investigated whether the numbers of IS481 and IS1001 copies vary in recently circulating isolates of the different Bordetella species. We used the polymerase chain reaction (PCR) method for screening the genome of circulating isolates and to prepare the fragments for sequencing. We used Southern blotting and quantitative real-time PCR for quantification of the numbers of IS. We found no significant diversity in the sequences of the IS harboured in the genomes of the Bordetella isolates screened, except for a 71-nucleotide deletion from IS1002 in B. bronchiseptica. The IS copy numbers in the genome of recently circulating isolates were similar to those in reference strains. Our results confirm that biological diagnosis targeting the IS481 and IS1001 elements are not specific and detect the species B. pertussis, B. holmesii and B. bronchiseptica (IS481), and B. parapertussis and B. bronchiseptica (IS1001).     

The use of TaqMan PCR assay for detection of Bordetella pertussis infection from clinical specimens

OBJECTIVE: The routine clinical laboratory detection of Bordetella pertussis is through culture, which can require 5 to 7 days for the bacteria to grow. Using a polymerase chain reaction (PCR) assay can shorten this detection time while increasing the sensitivity of detection with similar specificity. This study compared culture with TaqMan PCR for detection of B pertussis in clinical specimens and the turnaround time for each assay during the pertussis season. MATERIALS AND METHODS: Nasopharyngeal swabs in Regan-Lowe transport media were collected from 1556 persons who had symptoms of whooping cough or who had had contact with infected persons; the swabs were submitted for B pertussis detection during the pertussis season. A single nasopharyngeal swab from each patient was submitted for both culture and TaqMan PCR detection. Upon receipt of the specimens, the swabs were inoculated onto Regan-Lowe agar for culture and incubated for up to 7 days. The same swab was processed for PCR detection using TaqMan PCR assay. A second nested PCR was used on positive specimens for resolution purposes. The TaqMan PCR assay was performed 3 to 5 days a week, whereas the culture was performed 6 days a week. All specimens were processed on the same day or earliest possible working day for TaqMan or culture, and specimens queued for resolution by nested PCR were batched. RESULTS: There were a total of 275 PCR positives and 28 culture positives. After resolution with the second nested PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 97.4%, 87.6%, and 100% for TaqMan PCR and 11.6%, 100%, 100%, and 85.7% for culture. The average turnaround time for positive culture was 5.1 days, and the average turnaround time for PCR was 2.3 days. CONCLUSION: The TaqMan PCR assay has superior sensitivity and shorter turnaround time over culture because it can be finished within one working day. Furthermore, the same swab can be used for culture of the bacteria for antibiotic susceptibility testing. The early detection of pertussis using TaqMan PCR assay allows early intervention on the spread of the disease and the ability to culture the bacteria from the same swab, thereby eliminating the need for a second swab and allowing for detection of antibiotic resistance.     

Personality stability and its implications for clinical psychology

Over the past decade, a series of longitudinal studies have demonstrated that personality traits are stable in adulthood: There are no age-related shifts in mean levels, and individuals maintain very similar rank ordering on traits after intervals of up to 30 years. These findings should be of interest to clinicians because they point to important similarities between normal personality and personality disorders, facilitate research on the psychological processes that maintain both adaptive and maladaptive traits, serve as a reminder that current problems in functioning may be the expression of enduring personality patterns, and foster more realistic expectations about how much therapeutic change is possible.     

Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion

Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.     

Evaluation of a real-time PCR assay for detection of Bordetella pertussis and B. parapertussis in clinical samples

A real-time PCR assay based on the TaqMan technology was developed for the detection of Bordetella pertussis and B. parapertussis in clinical samples. The assay was evaluated with 182 specimens from 153 patients with and without symptoms of pertussis. The analytical sensitivity ranged from 0.1 to 10 cfu for B. pertussis and B. parapertussis, respectively, and diagnostic sensitivity was 94.1% when culture was used as a reference. No sample from a patient without symptoms of pertussis was positive in PCR. Twenty-four of 28 patients who were negative by culture and positive by PCR assay met the CDC clinical case definition for pertussis; the remaining four patients had paroxysms of shorter duration. Intra- and inter-assay variation were <5% and results were available within 4 h.     

Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)-PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD-PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD-PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.     

Neonatal diagnosis of Prader-Willi syndrome and its implications

Although Prader-Willi syndrome (PWS) patients usually first present with neonatal hypotonia and feeding difficulty, they later show hyperphagia, obesity and mental retardation. Since deletions of chromosomes 15q11-q13 are noted in most PWS patients cytogenetic analysis allows one to diagnose infants suspected of PWS with a greater certainty. We report on 5 hypotonic infants clinically suspected of PWS in the first 3 months of life, whose diagnosis was confirmed by cytogenetic studies showing monosomy of 15q11-q13. Early diagnosis of PWS can lead to prevention of obesity, but counseling of parents has been difficult. Although there are significant benefits to the early diagnosis of PWS, the cost-effectiveness and practicality of screening all hypotonic infants using high resolution cytogenetic analysis has been addressed systematically.     

Multitarget FISH analysis in the diagnosis of lung cancer

The aim of the present study was to explore the diagnostic usefulness of the multitarget fluorescence in situ hybridization (FISH) test, LAVysion (Vysis, Downers Grove, IL), for the detection of lung cancer cells in cytologic specimens. Specimens from bronchial washings, bronchial brushings, and transbronchial fine-needle aspirates (TBNAs) from 100 patients with suspected lung cancer and from a control group of 71 patients with nonneoplastic lung disorders were analyzed. FISH positivity was defined as more than 5 cells with gains of at least 2 chromosomes or gene loci. FISH significantly improved the sensitivity of bronchial brushings from 49% to 73%. The specificities of FISH and cytologic examination were 87% and 100%, respectively. In contrast, FISH provided no additional diagnostic information in TBNAs and bronchial washings. There was no increased prevalence of genetic changes in contralateral bronchial washings from patients with lung cancer compared with the control group. The quantity of previous smoking had no effect on the prevalence of chromosomal changes.     

Evaluation of PCR for Diagnosis of Bordetella pertussis and Bordetella parapertussis Infections

PCR, using primers PIp1 and PIp2, was evaluated for the detection of DNA from Bordetella pertussis in bacterial strains and in nasopharyngeal samples from patients with a cough lasting at least 7 days. The assay could detect DNA from 6 CFU of B. pertussis/10 μl of sample. Results of the PCR assay were compared with those of cultures, a determination of serum antibodies against pertussis toxin and filamentous hemagglutinin, and a clinical evaluation of 2,442 coughing episodes. The overall sensitivity of PCR was 65% (623 of 956), which was higher than the sensitivity of cultures (58%) (P < 0.001). Factors influencing the sensitivity of PCR were the interval between the onset of symptoms and sampling and the vaccination status of the patient. The specificity of PCR was 98% (1,451 of 1,486). The positive and negative predictive values were 95 and 81%, respectively. Parapertussis PCR, using primers BPPA and BPPZ, was positive in 11 of 18 culture-positive cases and was confirmed by serology in another 4 cases. In conclusion, PCR is a valuable complement to cultures and can probably replace cultures for diagnosis of B. pertussis and Bordetella parapertussis infections.     

Real-time PCR and its application to mumps rapid diagnosis

A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens.     

Comparison of real-time PCR and conventional hemi-nested PCR for the detection of Bordetella pertussis in nasopharyngeal samples

We directly compared a conventional hemi-nested PCR assay with a real-time (LightCycler) PCR assay for the detection of Bordetella pertussis in nasopharyngeal samples. Of the 152 samples tested, 49 (32%) were positive by first-round conventional PCR, 56 (37%) were positive by the hemi-nested PCR assay, and 39 (26%) were positive by the real-time assay. All samples testing positive with the real-time assay were also positive by the hemi-nested assay (both first- and second-round PCR), and all culture-positive samples tested positive by both PCR assays. These results suggest that the hemi-nested assay is more sensitive than the real-time assay for detecting B. pertussis .     

PCR for diagnosis of paracoccidioidomycosis

A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa. In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis.     

Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance

A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.     

Laboratory diagnosis of equine rabies and its implications for human postexposure prophylaxis

Laboratory diagnosis is essential to confirm suspected cases of equine rabies and to determine the medical care needed for human postexposure antirabies prophylaxis. Equine rabies transmitted by the vampire bat, Desmodus rotundus, has increased gradually in the State of S?o Paulo. The present study has several objectives, the most important being the evaluation of fluorescent antibody test (FAT) and virus-isolation laboratory tests performed with different equine nervous system tissues (cortical, hippocampus, cerebellar, brainstem and cervical medullar) to determine the tissue for which the two techniques have the highest sensitivity. Analysis by FAT of these five regions of the central nervous system (CNS) from 35 animals showed that there was a greater amount of viral antigen in the brainstem and cervical medullar tissues than in the hippocampus, cortical and cerebellar tissues. While there were no significant differences in the mortality rate of mice inoculated with suspension prepared from the different tissues, a trend towards higher mortality rate was detected with brainstem and cervical medullar tissues. Laboratory diagnosis was not affected by whether the animal had been vaccinated or not, or whether it had died following the natural course of the disease or as a result of euthanasia. Isolation of the rabies virus in equine salivary glands demonstrated the potential risk for humans exposed to infected animals.     

Real-time LightCycler PCR for detection and discrimination of Bordetella pertussis and Bordetella parapertussis

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.